Pharmaceutical composition for inducing an immune response in a human or animal

ABSTRACT

The present invention relates to a pharmaceutical composition for inducing an immune response in a human or animal, comprising dendritic cells loaded with at least five cancer/testis antigen and no lineage specific differentiation antigens or substantially no lineage specific differentiation antigens provided from at least one cancer cell line, as well as to isolated cell lines expressing a multiplicity of cancer testis antigens and no differentiation antigens, and to a method of inducing an immune response in a human or animal using the composition of the invention.

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition forinducing an immune response in a human or animal. In another aspect theinvention relates to a method for obtaining autologous dendritic cellsloaded with at least five cancer/testis antigen and no lineage-specificdifferentiation antigen or substantially no lineage-specificdifferentiation antigen. In another aspect the present invention relatesto an isolated melanoma cell line. In a further aspect the presentinvention relates to a use of the composition as an immunotherapeuticvaccine and a use of autologous dendritic cells as antigen presentingcells in a pharmaceutical composition or a vaccine. In an even furtheraspect the present invention relates to a method for inducing an immuneresponse in a human or animal.

BACKGROUND ART

Advanced cancers represent one of the major causes of human death. Noeffective methods of treatment have been suggested so far. Cancerimmunotherapy aims at destroying tumor cells by immunologicalmechanisms. Immunotherapy compared to conventional methods of cancertherapy like surgery, radiation, and chemotherapy, is much less toxic,and no serious complications have been described so far. In addition,immunotherapy has the potency to work at different stages of disease. Atinitial stages it could be a good supplementary treatment to surgicalremoval of primary tumors, aiming to prevent development of disseminateddisease. At advanced stages of disease it could be the only means oftreatment, as conventional methods are often ineffective.

The main function of the immune system is to identify and destroyforeign sub-stances (antigens) that invade the organism. The immunesystem is able to discriminate “self” from “non-self”, and under normalconditions only to develop an immune response against foreign or“non-self” antigens. Even though cancer cells originate from theorganisms own cells they are treated as foreign by the natural immunesystem. However, this natural immune response is not strong enough inorder to block the appearance and growth of the tumor. The task ofimmunotherapy is to increase the ability of the immune system torecognize tumor cells and to develop effective mechanisms of tumorelimination. The two main questions in any specific immunotherapy arewhich antigens to target and to find the optimal antigen presentation tothe immune system.

Tumor associated antigens (TAA) recognized by cytotoxic T lymphocytes(CTL) is the most efficient component to be targeted among knowneffector mechanisms in anti-tumor immunity. There are two major types ofTAA: unique antigens, present only in very few tumors and therefore notuseful as general targets, and shared or common TAA, present in manytumors. Three major groups of shared antigens are currently consideredas potential targets for immunotherapy and have all been shown to inducegeneration of cytotoxic T lymphocytes. The three groups are:Cancer/testis antigens (CT antigens), Tumor over-expressed antigens, andLineage-specific differentiation antigens.

CT antigens are encoded by cancer or germ line specific genes,representing one of the largest groups of shared tumor-associatedantigens. CT antigens were originally discovered in melanomas but havealso been found in many other human malignancies. Among normal tissuethey are only expressed in testis and in some cases in placenta. Normalcells expressing these antigens lack expression of MHC molecules andtherefore these antigens are normally not accessible for recognition byT lymphocytes.

This makes CT antigens very attractive targets for specific cancerimmunotherapy. Recent clinical trials have demonstrated tumor regressionin a significant number of melanomas and bladder cancer patients bytargeting of one specific CT antigen ((Nishiyama et al 2001, Clin.Cancer Res., v. 7, pp. 23-31; Thurner et al., 1999, J. Exp. Med., v.190, pp. 1669-1678)). CT antigen recognition by T cells has only beenreported for some CT antigens and the corresponding peptide epitopesdetermined. However, all CT antigens could be considered potentialtargets for immunotherapy. A correlation of the expression of MAGE-Aantigens and tumor progression has been found in a number ofmalignancies ((Brasseur et al., 1995, Int. J. Cancer, v. 63, pp.375-380; Eura et al., 1995, Int. J. Cancer, v. 64, pp. 304-308; Katanoet al., 1997, J. Surg. Oncol., v. 64, pp. 195-201; Patard et al., 1995,Int. J. Cancer, v. 64, pp. 60-64)). Another group of CT antigens, theMAGE-B antigens, shows a significantly lower tumor-specific expressionthan the MAGE-A antigens. A third group, the MAGE-C antigens, displaysan expression pattern that resembles the pattern of the MAGE-A antigens.No CTL response against MAGE-C antigens has been reported yet.

Several non-MAGE proteins with the characteristics of CT antigens havebeen described. One of them, NY-ESO-1 is one of the most immunogenictumor antigens identified to date. Clinical trials with peptideimmunization of melanoma patients demonstrated stabilization of diseaseand regression of some metastases in some patients ((Jager et al., 2000,Proc. Natl. Acad. Sci. U. S. A, v. 97, pp. 12198-12203)).

In contrast to CT antigens, tumor over-expressed antigens lack strictlytumor specific expression, as their expression could be detected in lowlevels in some normal tissue types other than testis. Development ofimmunotherapy to some of these antigens could be beneficial for cancerpatients, and presently such antigens like CEA, p53, HER-2/Neu, MUC-1and alpha-fetoprotein are being intensively investigated as possibletargets in clinical trials. The group of tumor over-expressed antigenshas only recently been used as targets in clinical trials, and thus dataon the efficiency of induction of therapeutic immune responses areabsent.

The lineage specific antigens, melanocyte differentiation antigens andprostate-associated antigens, have so far only been described for twotypes of human cancers: melanomas and prostate cancer. This group ofantigens is expressed both in normal differentiated tissue and in twotypes of human cancers. In normal differentiated tissue these antigensvery rarely induce an immune response, however, these proteins becomeimmunogenic in cancer cells, and in the case of melanomas, it ispossible to detect T-killer cells reactive against melanocytedifferentiation antigens. A prevailing number of clinical trailsdirected against melanomas or prostate cancer employ targeting ofdifferentiation antigens.

Among the groups of tumor associated antigens the most promising datausing TAA as targets for immunotherapy were obtained with some of theMAGE proteins. However, especially in case of melanomas, the therapeuticeffect was unstable, and some metastases continued to grow. Thesemetastases were usually negative for expression of the MAGE antigensused for immunization ((Thurner et al., 1999, J. Exp. Med., v. 190, pp.1669-1678)).

One possibility for inducing a polyvalent immune response is to employwhole tumor cells or material derived from whole cells. WO 9003183, U.S.Pat. No. 5,840,317, and U.S. Pat. No. 6,187,306 describes severalmelanoma cell based vaccine preparations. U.S. Pat. No. 4,108,983discloses a first generation melanoma vaccine derived from melanomacells lysed by a vaccinia virus.

U.S. Pat. No. 5,635,188 and U.S. Pat. No. 5,030,621 disclose a vaccineof cell surface antigens from melanoma cells that are shed into theculture medium and subsequently used as an anti-melanoma vaccine.Similarly, U.S. Pat. No. 5,484,596 discloses a method of cancer therapywherein irradiated tumor cells are injected into a human patient as avaccine. U.S. Pat. No. 6,187,306 relates to melanoma cell linesexpressing shared immunodominant melanoma antigens and methods of use.

An important aspect of any vaccine therapy is the way of vaccineadministration. In recent years it has been realized that the mostefficient way of antigen delivery to T cells, especially to naïve Tcells, is by way of dendritic cells. Dendritic cells (DC) are the mostefficient antigen presenting cells and DC based immunotherapy havealready been used in different settings for treatment of cancer ((Kugleret al., 2000, Nat. Med., v. 6, pp. 332-336; Nestle et al., 1998, Nature(Med.), v. 4, pp. 328-332; Thurner et al., 1999, J. Exp. Med., v. 190,pp. 1669-1678)) demonstrating high potency of this way of immunization.

One of the unique properties of DC is their ability to uptake exogenousproteins by endocytosis, which are then processed and presented aspeptide epitopes on their surface in conjunction with MHC class Iantigens. The antigen presenting dendritic cells can be recognized bycytotoxic T cells. This property is extremely important when tumor cellantigens are applied in form of tumor lysates or apoptotic bodies addedexogenously. High endocytic activity is believed to be associated withthe immature state of DC differentiation based on comparison of immatureand mature DC ((Sallusto et al., 1995, J. Exp. Med., v. 182, pp.389-400)). The possibility that differences in the endocytic activityamong immature DC could exist has never been considered.

WO 0127245 discloses a method of obtaining dendritic precursor cellsfrom peripheral blood by standard leukapheresis, buoyant densitygradient centrifugation and culture of the cells ex vivo in serum freemedium for 40 hours in the absence of exogenously added cytokines.

WO 0146389 relates to a method for generating dendritic cells fromleukapheresis products in closed systems, by using culture medium devoidof non-human proteins. Clinical grade cytokines (IL-4 and GM-CSF) areused in the culture medium and TAA are added for loading of the DCs.

Thurner et al., 1999, J. of Immunological Methods 223: 1-15, relates toa method for reproducible generation of large numbers of mature DCs fromCD14+ monocytes by a two step method where cytokines are added after day1.

The combination of dendritic cells with TAA's is also disclosed in WO0128583, which relates to an immunotherapeutic vaccine providing antigenpresenting cells mat have been pulsed with a disrupted cell preparationwhich includes cell membranes of cancer cells infected with recombinantvaccinia virus encoding at least one immunostimulating molecule. Alsoincluded is autologous DCs that presents a mixture of antigens frommelanoma cell lines infected with a recombinant vaccinia virus encodingIL-2. In WO 0129192 a method is disclosed for inducing a tumor specificimmune response in a patient, wherein antigen presenting cells from thepatient are incubated with dead cell portions possessing at least onetumor antigen and the resulting loaded antigen presenting cells areadministered to the patient.

Although the immunotherapeutic vaccines for treatment of cancer haveimproved over the recent years a need still exists for safer and moreefficient compositions for use in cancer immunotherapy. Such vaccinesshould be polyvalent targeting several CT antigens to avoid outgrowth ofantigen loss variants. They should be safer without the potential riskof targeting antigens expressed in normal tissue. They should beoptimized for the presentation and delivery of the antigens to the Tcells and also care should be taken to select the most efficient TAA'sas targets.

DISCLOSURE OF INVENTION

The present invention has solved these problems by careful selection ofthe melanoma cell lines to supply the most effective TAA's followed by asubsequent screening in order to avoid any antigens which potentiallycould be harmful to the patient. Also the antigen presenting cells havebeen optimized for their endocytic activity before loading the DCs withthe whole melanoma cell lysate.

In a first aspect the present invention relates to a pharmaceuticalcomposition for inducing an immune response in a human or animal,comprising dendritic cells presenting a multiplicity of cancer/testisantigens, wherein

a) at least five cancer/testis antigens and no lineage specificdifferentiation antigens or substantially no lineage specificdifferentiation antigens are presented by the dendritic cells,b) the cancer/testis antigens are provided from at least one cancer cellline expressing at least five different cancer/testis antigens and nolineage specific differentiation antigens or substantially no lineagespecific differentiation antigens, andc) the dendritic cells are immature (CD1a positive, CD14 negative, andCD83 negative) during loading of the cancer/testis antigens.

In a second aspect the present invention relates to a method forobtaining human or animal autologous dendritic cells loaded with atleast five cancer/testis antigens and no lineage specificdifferentiation antigens or substantially no lineage specificdifferentiation antigens, comprising the steps:

a) providing at least one cancer cell line expressing the at least fivecancer/testis antigens and no lineage specific differentiation antigensor substantially no lineage specific differentiation antigens,b) providing autologous dendritic cells from said human or animal,c) using a seeding density of monocytes between 5×10⁶-20×10⁶ cells per25 cm²,d) culturing said dendritic cells ex vivo in growth medium without anycytokines in an initial growth phase, followed by a second growth phasein medium comprising cytokines, ande) loading said dendritic cells from d) with the cancer/testis antigensobtained from a whole cell lysate of the at least one cancer cell linefrom a).

In a third aspect the present invention relates to an isolated melanomacell line, expressing at least five cancer/testis antigens and nomelanocyte differentiation antigens or substantially no melanocytedifferentiation antigens.

In a fourth aspect the present invention relates to a use of amultiplicity of cancer/testis antigens obtainable from an isolatedcancer cell line of claim 28 in a pharmaceutical composition or vaccineformulation.

In a fifth aspect the present invention relates to a use of dendriticcells as antigen presenting cells in a pharmaceutical composition or avaccine, and where the said dendritic cells are loaded with the antigensin their immature state at which point the dendritic cells are CD1apositive, CD14 negative, CD83 negative, wherein the dendritic cells havebeen cultured ex vivo in growth medium without any cytokines in aninitial growth phase, followed by a second growth phase in mediumcomprising cytokines before loading the dendritic cell with at leastfive cancer/testis antigen.

In a sixth aspect the present invention relates to a method for inducingan immune response in a human or animal comprising the steps:

a) providing at least one cancer cell line expressing at least fivecancer/testis antigens and no lineage specific differentiation antigensor substantially no lineage specific differentiation antigens,b) providing autologous dendritic cells from said human or animal,c) culturing said dendritic cells ex vivo in growth medium without anycytokines in an initial growth phase, followed by a second growth phasein medium comprising cytokines,d) loading said dendritic cells from c) with the cancer/testis antigensobtained from a whole cell lysate of the at least one cancer cell linefrom a), ande) administering said loaded dendritic cells from d) to said human oranimal.

BRIEF DESCRIPTION OF THE DRAWING(S)

The invention is explained in detail below with reference to thedrawing(s), in which

FIG. 1 shows sensitivity to lysis by gp100 and MART-1/Melan A-specificcytotoxic T lymphocyte (CTL) clones of a number of DDM-1 melanomaclones.

FIG. 2 shows expression of melanocyte differentiation antigens gp100 andMART-1/Melan A in three melanoma cell clones, DDM-1.7, DDM-1.13, andDDM-1.29 analyzed by RT-PCR.

FIG. 3 shows expression of melanocyte differentiation antigen gp100 inDDM-1.7 and DDM-1.29, cells determined by immunostaining.

FIG. 4 shows expression of MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A12, NY-ESO-1 and GAPDH in the three melanoma cell clones, DDM-1.7,DDM-1.13, and DDM-1.29 analyzed by RT-PCR analysis.

FIG. 5 shows expression of MAGE-A and NY-ESO-1 in DDM-1.13 cells aftertreatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine.

FIG. 6 shows uptake by endocytosis of fluorospheres by four cultures ofimmature dendritic cells.

FIG. 7 shows production of IFN-γ by a gp100-specific CTL clone afterinteraction with four dendritic cell cultures (same as in FIG. 6) loadedwith lysate of DDM-1.29 cells.

FIG. 8 shows cytolytic activity of immune lymphocytes of donor ANBIagainst melanoma cells, EBV-transformed B cells and K562 cells.

FIG. 9 shows cytolytic activity of immune lymphocytes of donor 19/00against breast and squamous cell carcinoma cell lines.

FIG. 10 shows expression of MAGE-A, and GAPDH genes in breast cancercell lines analyzed by RT-PCR analysis.

BEST MODE(S) FOR CARRYING OUT THE INVENTION

Prior to a discussion of the detailed embodiments of the invention, adefinition of specific terms related to the main aspects of theinvention is provided.

DEFINITIONS

Cytotoxic T-lymphocytes: Lymphocytes are small mononuclear white bloodcells present in lymphoid tissues and circulating blood and lymph. Thereare two main functional types, B-lymphocytes and T-lymphocytes, whichtake part in antigen specific immune reactions. T-lymphocytes (T cells)are a type of small antigen-specific lymphocyte originating in thymus(in mammals) and present in secondary lymphoid tissues (e.g. lymphnodes, spleen) and blood, and which are involved in cellular immunereactions and aiding the production of antibodies. T lymphocytes bearantigen-specific receptors on their surface and only react to foreignantigen presented to them on the surface of a cell. The main types of Tlymphocytes are: cytotoxic T cells which recognize and kill body cellsmat have become antigenically altered in some way (e.g. by virusinfection), helper T cells which are activated by foreign antigen on thesurface of antigen-presenting cells and in turn activate the appropriateB cells, suppressor T cells which are involved in suppressing immuneresponses and the general regulation of the immune system.

Cancer/testis antigens (CT-antigens): Antigens encoded by cancer or germline specific genes, representing one of the largest groups of sharedtumor-associated antigens. CT-antigens were originally discovered inmelanomas but have also been found in many other human malignancies.Among normal tissue they are only expressed in testis and in some casesin placenta. Normal cells expressing these antigens lack expression ofMHC molecules and therefore these antigens are normally not accessiblefor recognition by T lymphocytes.

Lineage specific differentiation antigens: A group of differentiationantigens, so far only described for melanomas and prostate cancer. Theseantigens are only immunogenic in cancer cells and the group comprisesthe melanocyte differentiation antigens gp100, Melan-A/MART-1,Tyrosinase, TRP-1, TRP-2, MC1R, AIM-1 and the prostate-associatedantigens PSA (prostate specific antigen), PSMA (prostate specificmembrane antigen), PAP (prostate associated phosphatase), and PSCA(prostate stem cell antigen).

Melanocyte differentiation antigens: A group of antigens expressed bothin normal differentiated melanocytes and in melanomas. In normaldifferentiated melanocytes these antigens very rarely induce an immuneresponse, however, these proteins become immunogenic in cancer cells,and in the case of melanomas, it is possible to detect T-killer cellsreactive against melanocyte differentiation antigens. The proteins arethought to be responsible for the synthesis of the pigment melanin.

Dendritic cells: Type of non-lymphocyte cell in some lymphoid tissues,which acts as an antigen-presenting cell by endocytosis of exogeneousproteins which are then processed and presented as epitopes on theirsurface in conjunction with MHC class I and II antigens. The antigenpresenting dendritic cells can be recognized by cytotoxic T cells andT-helper cells. The maturation state of the dendritic cells areimportant for their phagocytic/endocytic activity. Immature dendriticcells are the most efficient cells for loading antigens.

Immature dendritic cells: Dendritic cells in which the expression ofcertain cell markers CD1a, CD14 and CD83 is characterized by a highexpression of CD1a (more than 50% of DCs in the population are positivefor CD1a), no expression or low expression of CD14 (less than 15% of DCsin the population are positive for CD14) and low expression of CD83(less than 25% of DCs in the population are positive for CD83).

Exosomes: Exosomes are membrane vesicles of 30 to 100 nm in diameter, ofendocytic origin, and are produced and secreted in vitro by living cellsof diverse origin.

Cytokines: Immune system proteins that are biological responsemodifiers. They coordinate antibody and T-cell immune systeminteractions, and amplify immune reactivity. Cytokines include monokinessynthesised by macrophages and lymphokines produced by activated Tlymphocytes and natural killer cells. Monokines include interleukin(IL)-1, tumor necrosis factor (TNF), α- and β-interferon (IFN), andcolony-stimulating factors. Lymphokines include IL's, γ-IFN,granulocyte-macrophage colony-stimulating factor (GM-CSF), andlymphotoxin. Endothelial cells and fibroblasts and selected other celltypes may also synthesise cytokines. Examples of suitable cytokinesaccording to the invention include IL-4, GM-CSF, IL-13, IFN-γ, Flt-31,SCF, TNF-α

Melanoma: A malignant cancer or tumor of varying degree of severity,which tend to spread or metastasize in advanced stages of the disease.

Loading dendritic cells: The uptake of exogeneous proteins byendocytosis and the presentation of peptide epitopes on the surface ofthe dendritic cell. Sometimes also referred to as “pulsing”.

The term “substantially no lineage specific/melanocyte differentiationantigens” in connection with the present invention means that thecancer/melanoma cell line to be used for preparing a whole cell lysateshould not express lineage specific/melanocyte differentiation antigensin amounts that will result in stimulation of an immune response againstthe lineage specific/melanocyte differentiation antigen. In practice,this means that cancer/melanoma cells are insensitive to lysis bycytotoxic T lymphocytes specific against lineage specific/melanocytedifferentiation antigens (lysis is less than 10%, particularly less than5%, and more particularly less than 2% in a 4-hours cytotoxicity test),and only 1-2% of cells are positively stained by antibodies againstlineage specific/melanocyte differentiation antigens. In addition, theamount of RNA transcripts from the genes encoding the lineagespecific/melanocyte differentiation antigens is at least about 100 foldlower in these cell lines as compared to highly sensitive cell lines, asdetermined by semi-quantitative RT-PCR.

Immune response: A selective response mounted by the immune system ofvertebrates in which specific antibodies and/or cytotoxic cells areproduced against invading microorganisms, parasites, transplanted tissueand many other substances which are recognized as foreign by the body(antigens). The production of antibodies circulating in the blood isknown as a humoral immune response, the production of cytotoxic cells asa cell-mediated or cellular immune response.

Immunotherapeutic vaccine: A vaccine administered to treat and/orprevent further progression of a disease in a host already diagnosedwith the disease.

Autologous cells: Cells that are an individual's own cells.

Allogeneic: Genetically different, but of the same species.

Antigen presenting cell: Specialized lymphoid cell such as dendriticcells, B cells and monocytic cells, which are capable of inducing T cellactivation.

Monocyte: Phagocytic white blood cell related to macrophages. Monocytesrepresent another type of antigen presenting cells, which mainlyre-activate previously sensitized cytotoxic T lymphocytes.

Precursor dendritic cells: CD14+ monocytes present in peripheral bloodor CD34+cells present in bone marrow or in peripheral blood (especiallyafter mobilization).

Immunodominant: Antigen present in mixture with other antigenspredominantly stimulates immune response against itself.

The present invention relates to an improved therapeutic composition tobe used as e.g. an immunotherapeutic vaccine. The most efficient way ofantigen delivery to T cells, especially to naïve T cells, is by way ofautologous dendritic cells. Several different tumor associated antigensfrom either of the three groups: CT antigens, tumor over-expressedantigens, and lineage specific antigens have been used in clinicaltrials and so far the most promising results have been seen withantigens from the group of cancer/testis antigens.

Tumor over-expressed antigens, in contrast to CT antigens, lack strictlytumor specific expression, as their expression could be detected in somenormal tissues other than testis, albeit at significantly lower levelsthan in tumor cells. Such distribution prevents the development ofstrong tumor rejection immunity (due to elimination of highly reactive Tlymphocytes in the course of tolerance induction) and in case a responsecould be generated, the potential risk of developing autoimmunity willbe high. The group comprises a large number of antigens and several haverecently been targets in clinical trials.

The same restrictions as mentioned above also apply to the lineagespecific antigens, which group includes the differentiation antigens.This group of antigens is also expressed in corresponding normaldifferentiated tissue, and in healthy individuals they are very seldominducing immune attack due to the tolerance to “self” proteins. Forunknown reasons, these normal proteins become immunogenic in cancercells, and in case of melanomas, T-killer cells reactive againstmelanocyte differentiation antigens could easily be detected inpatients, but not in healthy individuals. A prevailing number ofclinical trails directed against melanomas or prostate cancer employtargeting of differentiation antigens.

It is an object of the present invention to specifically avoid thepresence of over-expressed and lineage-specific differentiation antigensin antigen mixture used for immunization. In the case of melanomas celllines, the group of over-expressed antigens, are extremely seldom ableto induce an immune response, therefore excluding a need for negativeselection for this group of antigens. Regarding melanocytedifferentiation antigens they should not be present, but if present theamount should not be sufficient to result in stimulation of an immuneresponse against them (in practice this means that the amount of RNAtranscripts from the genes encoding the melanocyte differentiationantigens is at least about 100 fold lower in the cell lines of theinvention as compared to highly sensitive cell lines, as determined bysemi-quantitative RT-PCR). The presence of such immunodominant proteinsshould be avoided and particularly the proteins gp100, Melan A/MART-1and tyrosinase should not be present.

It is also an object of the present invention to provide cell linesexpressing at least three, particularly at least five CT antigen andmore particularly as many CT antigens as possible, which should beimmunodominant. Surprisingly such cell lines could be provided frommelanoma patients, which after removal of the stage III or stage 1Vtumor have had a long disease-free period (more than five years),indicative of immunogenicity of the tumor cells in vivo. Individualsubclones from such cell lines should subsequently be screened for theabsence of any melanocyte differentiation antigens, particularly gp100,Melan A/MART-1 and tyrosinase.

In addition to melanoma immunotherapy the present invention alsoprovides a method for inducing an immune response in a human or animalfor other types of cancer. The requirements are that the antigenstargeted are shared antigens and pre-sent in other types ofmalignancies, predominantly in solid tumors. Particularly these types ofcancer may comprise colorectal carcinoma, pancreatic cancer, breastcancer, ovarian cancer, prostate cancer, squamous cell carcinoma, sweatgland carcinoma, renal cell carcinoma, hepatoma, cervical cancer, lungcarcinoma, small cell lung carcinoma or bladder carcinoma.

In one embodiment the present invention therefore relates to apharmaceutical composition for inducing an immune response in a human oranimal, comprising dendritic cells presenting a multiplicity ofcancer/testis antigens, wherein

a) at least five cancer/testis antigens and no lineage specificdifferentiation antigens or substantially no lineage specificdifferentiation antigens are presented by the dendritic cells,b) the cancer/testis antigens are provided from at least one cancer cellline expressing at least five different cancer/testis antigens and nolineage specific differentiation antigens or substantially no lineagespecific differentiation antigens, andc) the dendritic cells are immature (CD1a positive, CD14 negative, andCD83 negative) during loading of the cancer/testis antigens.

Advantageously the loaded dendritic cells may subsequently be matured bythe addition of maturation factors.

Dendritic cells are the most efficient antigen presenting cells asdiscussed above, and it is another objective of the present invention toprovide dendritic cells and particularly autologous dendritic cellsgenerated from CD14+ monocytes isolated from peripheral blood or CD34+cells from peripheral blood or bone marrow, which have been optimizedwith respect to their endocytic/phagocytic activity and CD1a expression.This activity is believed to relate to dendritic cells in their immaturestate. In order to obtain such activated cells most reports haveemployed the “GM-CSF+IL-4 method” by Sallusto and Lanzavecchia, (1994,J. Exp. Med. 179: 1109). Other suitable cytokines include IL-4, GM-CSF,IL-13, IFN-γ, Flt-31, SCF, TNF-α(Alters et al., 1999, J. Immunother., v.22, pp. 229-236), in particular in relation to the pre-sent inventionthe cytokines are selected from GM-CSF and IL-4.

Surprisingly we have now found, as shown in Example 2 below, that thismethod can be further optimized and that culture ex vivo in growthmedium without any cytokines in an initial growth phase, followed by asecond growth phase in fresh medium comprising cytokines before loadingthe dendritic cells results in DCs with increased endocytic activity.The initial growth phase according to the present invention is from 6-48hours, particularly from 12-34 hours, and more particularly from 20-28hours.

Previously it was believed that the endocytic activity of all immaturedendritic cells was equally good irrespective of the culture method. Nowwe have discovered that by applying a reported method for the productionof stable mature dendritic cells and loading the dendritic cells intheir immature state before addition of maturation factors, it ispossible to obtain immature dendritic cells, which have been optimizedin respect of their endocytic activity.

In another embodiment the present invention therefore relates to apharmaceutical composition as stated above, wherein the dendritic cellshave been cultured ex vivo in growth medium without any cytokines in aninitial growth phase, followed by a second growth phase in mediumcomprising cytokines before loading the dendritic cells with at leastone cancer/testis antigen.

Since a multiplicity of CT-antigens should be provided and particularlymore than five CT-antigens, the present invention in a furtherembodiment relates to a pharmaceutical composition as described above,wherein the at least five cancer/testis antigen are provided from awhole cell lysate of the at least one cancer cell line expressing nolineage specific differentiation antigens or substantially no lineagespecific differentiation antigens. A whole cell lysate can be obtainedin several ways from cells, like e.g. tumor cells or other cell types,by disrupting the cells, e.g. by several cycles of freezing and thawing.In the cell lysate, which comprises the soluble material, normallyparticles are removed by centrifugation and/or filtration.

In one particular embodiment the present invention relates to apharmaceutical composition as described above wherein the cancer cellline is a melanoma cell line, and the lineage specific differentiationantigens are melanocyte differentiation antigens.

In a further embodiment the melanocyte differentiation antigens comprisegp100, Melan A/Mart-1, and tyrosinase.

Dendritic cells cultured ex vivo according to the present invention willbe optimized in respect of their endocytic activity and CD1a expression,and they can advantageously be applied for antigen presentation in anycomposition or vaccine for use in immunotherapy. Accordingly in afurther embodiment the present invention relates to a use of autologousdendritic cells as antigen presenting cells in a pharmaceuticalcomposition or a vaccine, wherein said autologous dendritic cells havebeen cultured ex vivo in growth medium without any cytokines in aninitial growth phase, followed by a second growth phase in mediumcomprising cytokines before loading the dendritic cell in their immaturestate with at least five cancer/testis antigen.

Other ways of obtaining dendritic cells presenting the antigensaccording to the invention, e.g. by whole cell fusion, would also bepossible. This could be accomplished by fusion of dendritic cells withthe cells lines according to the invention.

In a further embodiment antigen presentation is performed by the use ofexosomes. Exosomes are small membrane vesicles of endocytic origin thatare secreted by most cells in culture and have recently been describedin antigen-presenting cells and they are capable of stimulating immuneresponses in vivo (Théry et al., 2002, Nature Reviews Immunology2:569-579).

In a still further embodiment the present invention relates to a methodfor obtaining autologous dendritic cells loaded with at least fivecancer/testis antigen and no lineage specific differentiation antigensor substantially no lineage specific differentiation antigens,comprising the steps:

a) providing at least one cancer cell line expressing at least fivecancer/testis antigens and no lineage specific differentiation antigensor substantially no lineage specific differentiation antigens,b) providing autologous dendritic cells from said human or animal,c) culturing said dendritic cells ex vivo in growth medium without anycytokines in an initial growth phase, followed by a second growth phasein medium comprising cytokines,d) loading said dendritic cells from c) with the cancer/testis antigensobtained from a whole cell lysate of the at least one cancer cell linefrom a).

Advantageously the dendritic cells may subsequently be matured afterstep d) by the addition of maturation factors such as e.g. IL-1β, IL-6,TNF-α; and PGE2.

In another embodiment the present invention relates to a pharmaceuticalcomposition obtainable by performing the steps a) through d) above,followed by a maturation step.

Employing the known methods for the generation of dendritic cells frompopulations of mononuclear cells good yields of dendritic cells havebeen difficult to obtain. The average yield of dendritic cells from astarting population of mononuclear cells has previously been reported tobe about 5% (Marovitch et al., 2002, J. Infect. Dis. 186: 1242-1252). Inorder to get 50×10⁶ dendritic cells, which is required for one completecycle of vaccination, it is necessary to start with 10⁹ mononuclearcells.

Assuming that the cell concentration during adsorption is usually5×10⁶/ml, this amount of cells will require 0.2 l of medium for theadsorption step, and the same amount of medium for further cellcultivation. About 60 μg of GM-CSF and 30 μg of IL-4 will be requiredfor the generation of dendritic cells, as well as large amount of tissueculture plastic ware. This will result in a net price per vaccine whichwill be rather high. In addition, in order to isolate 109 mononuclearcells from the blood, up to 1 L of blood will be required, which ishardly possible to draw from a patient even if performed in two drawingsseparated significantly in time considering the poor health of thepatient.

One possibility is to employ leukapheresis that permits the isolation ofsignificant number of mononuclear cells. This procedure is often used toproduce a large number of dendritic cells (see e.g. Thurner et al. 1999,J. Immunol. Methods 223: 1-15). The procedure of leukopheresis is,however, highly time-consuming and costly, which in turn will increasethe total price of vaccine production. In addition, only one patient canbe processed on a single leukapheresis equipment. This will limit theproductive capacity of manufacturing procedures.

Alternatively, the efficiency of the generation of dendritic cells frommonocytes could be increased. For example Tuyaerts et al. 2002, (J.Immunol. Methods 264: 135-151) have reported the adaptation of themethod of dendritic cell production for Nunc Cell Factories, andreported a yield of dendritic cells up to 40% of the starting number ofmonocytes (or 13% of the starting number of mononuclear cells, asmonocytes represent on average one third of the mononuclear cells). Thegenerated dendritic cells, however, were lacking the expression of theCD1a marker, which may indicate their incomplete differentiation. Sinceexpression of CD1a is essential for the purpose of the present inventionthis method cannot be employed for our purpose.

Therefore, we have instead optimized the procedure for generation ofimmature dendritic cells from blood monocytes, closely monitoring theincreasing efficiency of the monocyte transformation into dendriticcells, as well as the generation of fully competent immature dendriticcells. As a read out system for the generation of competent immaturedendritic cells, we have selected following criteria: high (more than50%) expression of CD1a marker, no or low (less than 15%) expression ofCD14 and low (less than 25%) expression of CD83.

The results of our optimization as described in the examples below hasdemonstrated the importance of the delayed addition of cytokines on theexpression of especially the CD1a marker.

Furthermore our studies have revealed that it is possible to obtain ayield of about 50% immature dendritic cells with high expression of CD1amarker generated from monocytes, by controlling the initialconcentration of monocytes in the seeding population of mononuclearcells.

In a further aspect the present invention therefore relates to a methodfor optimizing the yield of dendritic cells generated from a sample ofmononuclear cells in which method the seeding density of monocytes isbetween 5×10⁶-20×10⁶ cells per 25 cm² in 6-8 ml of medium. When using aT25 flask, this means a starting number of monocytes of between5×10⁶-20×10⁶ cells. In a particular embodiment the density is between6×10⁶-15×10⁶ cells per 25 cm², and more particularly between8×10⁶-12×10⁶ cells per 25 cm².

In a particular embodiment at least two allogeneic melanoma cell linesare provided in step a). The number of allogeneic cell lines depends onthe number of suitable subclones that have been isolated and screenedfor the absence of melanocyte differentiation antigens. The more celllines provided the better are the chances that several immunodominantcancer/testis antigens will be represented in the whole cell lysate. Inone embodiment the at least five CT-antigens are provided from at leasttwo allogeneic cell lines. In a particular embodiment the allogeneiccell lines are selected from DDM-1.7 (ECACC 01112339) or DDM-1.13 (ECACC01112338) (Both cell lines deposited at the European Collection ofAnimal Cell Cultures, CAME, GB-Salisbury, Wiltshire SP4 0JG, UnitedKingdom on 23 Nov. 2001).

Another aspect of the invention relates to the above particular celllines and also to other cell lines expressing at least five CT antigensand no melanocyte differentiation antigens or substantially nomelanocyte differentiation antigens. Accordingly the present inventionrelates to an isolated melanoma cell line, expressing at least fivecancer/testis antigen and no melanocyte differentiation antigens orsubstantially no melanocyte differentiation antigens, and particularlyto the isolated cell lines DDM-1.7 (ECACC 01112339) or DDM-1.13 (ECACC01112338). Accordingly the present invention also relates to apharmaceutical composition or a method of the present invention whereinat least one melanoma cell line is selected from the allogeneic celllines DDM-1.7 (ECACC 01112339) or DDM-1.13 (ECACC 01112338).

The pharmaceutical composition when administered to a human or animal,will induce an immune response in said human or animal resulting in thestimulation of the production of cytotoxic T lymphocytes in the human oranimal, and in a further object the invention therefore relates to amethod for inducing an immune response in a human or animal comprisingthe steps:

a) providing at least one cancer cell line expressing cancer/testisantigens and no lineage specific differentiation antigens orsubstantially no lineage specific differentiation antigens,b) providing autologous dendritic cells from said human or animal,c) culturing said dendritic cells ex vivo in growth medium without anycytokines in an initial growth phase, followed by a second growth phasein medium comprising cytokines,d) loading said dendritic cells from c) with the cancer/testis antigensobtained from a whole cell lysate of the at least one cancer cell linefrom a),

e) administering said loaded dendritic cells from d) to said human oranimal.

In a particular embodiment of the above method the cancer cell line is amelanocyte cell line, and the lineage specific differentiation antigensare melanocyte differentiation antigens.

In some cases it will be difficult to provide sufficient amounts ofautologous dendritic cells from the patient. In this case it is possibleto administer substances that will induce mobilization of CD14+monocytes prior to step b). Said substances comprise G-CSF and/orGM-CSF.

The dendritic cell precursors, CD14+ monocytes or CD34+ cells, can beobtained from a blood sample, from peripheral blood. It is also possiblebut not necessary to start from apheresis cells.

Also it is contemplated that the method according to the presentinvention further comprises the step of administering to the human oranimal, a substance that induces activation of T lymphocytes after stepe). This could be accomplished by administration of e.g. IL-2 or IL-12.

The present invention also contemplates the use of agents that mayincrease the level of expression of the cancer/testis antigens beforepreparing the whole cell lysate of the at least one melanoma cell line.DNA methylation has been suggested to influence the expression level ofsome testis-specific genes. It has been demonstrated that thedemethylating agent 5-aza-2′deoxycytidine (5azaCdR) can induce theexpression of the MAGE-A1 gene in MAGE-A1-negative melanoma cells ((DeSmet et al., 1996, Proc. Natl. Acad. Sci. U.S.A., v. 93, pp. 7149-7153;Weber et al., 1994, Cancer Res., v. 54, pp. 1766-1771)). 5azaCdR is acytosine analogue that acts as a suicide substrate for DNAmethyltransferase when incorporated into DNA at the target site for DNAmethylation, CpG dinucleotides. Demethylation in eukaryotic cellsnormally leads to increased gene expression in vivo. It has beenproposed that MAGE-A1 activation results from the demethylation of thepromoter region, following an overall demethylation process, whichoccurs in many tumors. The activation effect of 5azaCdR on geneexpression has also been shown for other members of the MAGE family((Lucas et al., 1998, Cancer Res., v. 58, pp. 743-752; Lurquin et al.,1997, Genomics, v. 46, pp. 397-408)) and for the GAGE ((De Backer et al,1999, Cancer Res., v. 59, pp. 3157-3165; Li et al., 1996, Clin. CancerRes., v. 2, pp. 1619-1625)) and LAGE ((Li et al., 1996, Clin. CancerRes., v. 2, pp. 1619-1625)) gene families. The role of demethylation inthe expression of MAGE genes in tumor cells is supported by the factthat the expression of many other testis-specific genes, whose presencewas not detected in tumors, was not upregulated by 5azaCdR treatment((De Smet et al., 1997, Biochem. Biophys. Res. Commun., v. 241, pp.653-657)), and among the MAGE-B genes the tumor expression have beendetected only for those which are activated by 5azaCdR treatment((Lurquin et al., 1997, Genomics, v. 46, pp. 397-408)). In addition, agood correlation of the demethylation of CpG sites in the promoterregion of the MAGE-A1 gene and the expression of the gene has beenobserved ((De Smet et al., 1999, Mol. Cell Biol., v. 19, pp.7327-7335)).

In one embodiment up-regulation of expression of the CT antigens couldtherefore be done by demethylation of the DNA encoding the CT antigens.In particular this demethylation could be induced by treatment with5azaCdR. Yet another way of upregulation of CT antigen expression couldbe inhibition of histone deacetylation. These two types of treatmentcould be used either separately or in combination. Such treatment may beemployed only if cancer/testis antigens remain immunodominant.

Many CT antigens can be grouped into subfamilies that include severalmembers (see Table 1). They are the MAGE-A, MAGE-B, MAGE-C, GAGE, LAGEand SSX subfamilies. For the other antigens only one individual memberhas been discovered so far. These are the BAGE, SCP-1, TSP50, TRAG-3,SAGE, IL13R alpha, CT9 and CTp11 antigens. All CT antigens could for thepurpose of the present invention be considered as potential targets forimmunotherapy.

TABLE 1 Human cancer/testis antigens Family Members MAGE-A MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12 MAGE-B MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5,MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17 MAGE-C MAGE-C1, MAGE-C2, MAGE-C3,MAGE-C4, GAGE GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7,GAGE-8 PAGE-1, PAGE-2, PAGE-3, PAGE-4 XAGE-1, XAGE-2, XAGE-3 LAGELAGE-1a, LAGE-1b, Ny-ESO-1 SSX SSX-1, SSX-2, SSX-3, SSX-4, SSX-5Separate BAGE, SCP-1, TSP50, TRAG-3, SAGE, IL13R alpha, CT9, members:CTp11

One of the largest groups of CT antigens is the group of MAGE proteins,comprising three families, MAGE-A, MAGE-B and MAGE-C.

The MAGE-A genes represent a family of 15 closely related genes locatedon the long arm of chromosome X (region Xq28) ((Chomez et al., 2001,Cancer Res, v. 61, pp. 5544-5551; De Plaen et al., 1994, Immunogenetics,v. 40, pp. 360-369)), including the first identified gene encoding theantigen MAGE-A1 (previously designated MAGE-1) ((van der Bruggen et al.,1991, Science, v. 254, pp. 1643-1647)). In the majority of theinvestigated tumors only the expression of the MAGE-A1, -A2, -A3, -A4,-A6 and -A 12 genes has been demonstrated. Recently the expression ofother MAGE-A genes, including MAGE-A11 ((Jurk et al., 1998, Int. J.Cancer, v. 75, pp. 762-766)), MAGE-A10 ((Huang et al., 1999, J.Immunol., v. 162, pp. 6849-6854)), MAGE-A5, MAGE-A8 and MAGE-A9((Serrano et al., 1999, Int. J. Cancer, v. 83, pp. 664-669)) has alsobeen detected in several tumors.

The ability to present peptide epitopes recognized by cytotoxic Tlymphocytes (CTL) has been shown for MAGE-A1 ((van der Bruggen et al.,1991, Science, v. 254, pp. 1643-1647)), MAGE-A2 ((Visseren et al., 1997,Int. J. Cancer, v. 73, pp. 125-130)), MAGE-A3 ((Gaugler et al., 1994, J.Exp. Med., v. 179, pp. 921-930)), MAGE-A4 ((Duffour et al., 1999, Eur.J. Immunol., v. 29, pp. 3329-3337)), MAGEA6 ((Tanzarella et al., 1999,Cancer Res., v. 59, pp. 2668-2674)), MAGE-A10 ((Huang et al., 1999, J.Immunol., v. 162, pp. 6849-6854)) and MAGE-A 12 ((PaneUi et al., 2000,J. Immunol., v. 164, pp. 4382-4392)). T-helper cells can also recognizeMAGE antigens, and corresponding epitopes of MAGE-A1 and MAGE-A3antigens have been identified ((Chaux et al., 1999, J. Exp. Med, v. 189,pp. 767-778; Chaux et al., 2001, Eur J Immunol, v. 31, pp. 1910-1916;Manici et al., 1999, J. Exp. Med., v. 189, pp. 871-876)).

MAGE expression has been detected in many types of human malignancies.Cutaneous melanomas have the highest level of MAGE expression (up to 65%for MAGE-A3) ((De Plaen et al., 1994, Immunogenetics, v. 40, pp.360-369)), while ocular melanomas usually are negative for MAGEexpression ((Mulcahy et al., 1996, Int. J. Cancer, v. 66, pp. 738-742)).To a lesser extent MAGE antigens are expressed in other types of tumorssuch as mammary carcinomas, head and neck tumors, lung carcinomas,sarcomas and bladder carcinomas (for review see (van Pel et al., 1995,Immunol. Rev., v. 145, pp. 229-250)). A high expression of MAGE-A1 (80%)was found in hepatocarcinomas ((Yamashita et al., 1996, Hepatology, v.24, pp. 1437-1440)). MAGE-A4, in contrast to other MAGE-A antigens, isexpressed in significant proportions of lymphomas, including Hodgkin'slymphomas, where its expression is restricted to Reed-Sternberg cells((Chambost et al., 2000, Blood, v. 95, pp. 3530-3533)). When a tumorsample is found to be positive for MAGE-A4, the gene is usuallyexpressed at very high levels.

The MAGE-B genes represent a family of 17 genes located in the regionsp21.3 and p22 of the X chromosome, with 8 of them being pseudogenes((Chomez et al., 2001, Cancer Res, v. 61, pp. 5544-5551; Lucas et al,2000, Int. J. Cancer, v. 87, pp. 55-60; Lurquin et al., 1997, Genomics,v. 46, pp. 397-408)). Only two genes, MAGE-B1 and MAGE-B2, are expressedin a significant fraction of tumors of various histological types. Theexpression of MAGE-B5 and MAGE-B6 was detected in a limited number oftumor samples ((Lucas et al., 2000, Int. J. Cancer, v. 87, pp. 55-60)).

The seven members of the MAGE-C family are located in the Xq26-q27region. The MAGE-C1 gene has been identified by analysis of theselective gene expression in testis and melanomas ((Lucas et al., 1998,Cancer Res., v. 58, pp. 743-752)). Its expression pattern stronglyresembles the expression pattern of the MAGE-A genes. Another gene CT7((Chen et al., 1998, Proc. Natl. Acad. Sci. U.S.A., v. 95, pp.6919-6923)), probably represent a different MAGE-C1 allele. MAGE-C2/CT10is localized in the Xq27 region but in contrast to MAGE-C1, this proteinhas no repetitive portion. The third and forth members, MAGE-C3 andMAGE-C4, were identified by database searching ((Chomez et al., 2001,Cancer Res, v. 61, pp. 5544-5551; Lucas et al., 2000, Int. J. Cancer, v.87, pp. 55-60)).

Several non-MAGE proteins with characteristics of cancer/testis antigenshave been described. One of these was the BAGE antigen ((Boël et al.,1995, Immunity, v. 2, pp. 167-175)). Its pattern of expression in tumorsamples is very similar to the expression pattern of the MAGE antigens,with an overall lower frequency of expression (22% in melanomas, 15% inbladder carcinomas, 10% in mammary carcinomas and 8% in head and necksquamous cell carcinomas). As for the MAGE antigens, the expression ofBAGE correlates with the stage of tumor progression. The BAGE antigencould be recognized by CTL's, and antigenic peptide epitopes wereidentified.

An additional antigen was identified as a HLA-Cw6-restricted epitopeencoded by the GAGE-1 gene ((Van den Eynde et al., 1995, J. Exp. Med.,v. 182, pp. 689-698)). This gene belongs to a large family of genes,including the GAGE-1-GAGES-8 genes ((Chen et al., 1998, J. Biol. Chem.,v. 273, pp. 17618-17625; De Backer et al., 1999, Cancer Res., v. 59, pp.3157-3165; Van den Eynde et al., 1995, J. Exp. Med., v. 182, pp.689-698)), the PAGE-1-PAGE-4 genes ((Brinkmann et al., 1998, Proc. Natl.Acad. Sci. U.S.A., v. 95, pp. 10757-10762; Chen et al., 1998, J. Biol.Chem., v. 273, pp. 17618-17625)), and the XAGE-1-XAGE-3 genes((Brinkmann et al., 1999, Cancer Res., v. 59, pp. 1445-1448)). The twogenes of the GAGE family that encode a protein, GAGE-1 and GAGE-2, areexpressed in a significant proportion of melanomas (24%), sarcomas(25%), non-small lung cancers (19%), head and neck tumors (19%), andbladder tumors (12%).

Several CT antigens have been identified recently using the SEREX method(serological expression cloning of recombinant cDNA libraries of humantumors) ((Sahin et al., 1995, Proc. Natl. Acad. Sci. U.S.A., v. 92, pp.11810-11813)). One of them, NY-ESO-1, encoded by the CTAG gene ((Chen etal., 1997, Cytogenet. Cell Genet., v. 79, pp. 237-240)), was expressedin 23 of 67 melanoma specimens, 10 of 33 breast cancers, 4 of 16prostate cancers, 4 of 5 bladder cancers, as well as a proportion ofother tumor types, but only in 2 of 11 cultured melanoma cell lines((Chen et al., 1997, Proc. Natl. Acad. Sci. U.S.A., v. 94, pp.1914-1918)). In a melanoma patient, the CTL response was restricted byHLA-A2, and three peptides recognized by a melanoma-specific CTL linehave been identified. This antigen was also found to induce aHLA-A31-restricted CTL response in one melanoma patient ((Wang et al.,1998, J. Immunol., v. 161, pp. 3596-3606)). In addition, MHC class IIrestriction by CD4+ T lymphocytes has been described, withidentification of three peptide epitopes ((Jäger et al., 2000, J. Exp.Med., v. 191, pp. 625-630)). A gene homologous to CTAG, has recentlybeen described. This gene, LAGE-1 ((Lethé et al., 1998, Int. J. Cancer,v. 76, pp. 903-908)) has a distribution in different tumors similar toNY-ESO-1. Both genes are located in the q28 band of the X chromosome,close to the MAGE genes ((Lethé et al., 1998, Int. J. Cancer, v. 76, pp.903-908)). NY-ESO-1 is one of the most immunogenic tumor antigensidentified to date.

Accordingly in a further embodiment of the present invention thecancer/testis antigens comprise antigens selected from the MAGE-A,MAGE-B, MAGE-C, GAGE, LAGE, SSX subfamilies.

In a particular embodiment the CT antigens comprise antigens selectedfrom MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A8,MAGE-A9, MAGE-A 10, MAGE-A11, MAGE-A 12, MAGE-B1, MAGE-B2, MAGE-B3,MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, BAGE, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5,GAGE-6, GAGE-7, GAGE-8, NY-ESO-1, LAGE, PAGE-1, PAGE-2, PAGE-3, PAGE-4,XAGE-1, XAGE-2, XAGE-3, SSX-1, SSX-2, SSX-3, SSX-4, SSX-5. And inanother particular embodiment the CT antigens comprise antigens selectedfrom SCP-1, TSP-50, TRAG-3, SAGE, IL-13R alpha, CTp11.

In a still further particular embodiment the CT antigens compriseantigens selected from MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10,MAGE-A12, and NY-ESO-1.

The above antigens represent antigens that are considered to beparticularly useful in the context of the present invention, however,other antigens not specifically mentioned could also be used as long asa multiplicity of cancer/testis antigens are used, and particularly atleast five, more particularly at least 6, and even more particularly atleast seven CT antigens.

A further embodiment of the present invention relates to a use of theabove composition as an immunotherapeutic vaccine for the treatment ofcancer.

One condition for employment of melanoma cell-based compositions orvaccines is the presence in a host of a malignancy at advanced stages ofdisease. Another condition could be presence of primary tumor, and inthis case the aim of treatment is not only to induce rejection of theprimary tumor, but also to prevent development of metastases, since agroup of CT antigens is predominantly expressed in metastases. Yetanother condition could be removal of primary or metastatic tumor byother means (surgery, irradiation), and in this case the aim oftreatment is the prevention of tumor recurrence.

Tumors that express several CT antigens have higher chances of beingrejected or restricted in growth than tumors that have no or only one CTantigen. Therefore, determination of expression of CT antigens in tumorbiopsy may be of significance in predicting the effectiveness ofemployment of a universal melanoma cell-based vaccine.

EXAMPLES

In the following the present invention will be further illustrated byway of several non-limiting embodiments.

Example 1 Isolation of Melanocyte Differentiation Antigen DeficientClones of the DDM-1 Melanoma Cell Line

DDM-1 melanoma cell lines obtained from a patient with a long diseasefree period. DDM-1 melanoma cells were cloned, and 8 clones werescreened and found to be negative for the expression of tyrosinase.Tyrosinase is one of the known melanocyte differentiation antigens.These clones (DDM-1.5, DDM-1.6, DDM-1.7, DDM-1.10, DDM-1.11, DDM-1.13,DDM-1.24, and DDM-1.25) were further tested for the expression of twoimmunodominant melanocyte differentiation antigens, gp100 and MelanA/MART-1.

Melanoma cells were routinely cultured in T75 tissue culture flasks(Nunc) in 20 ml of RPMI 1640 medium (BioWhittaker) supplemented with 10%of foetal calf serum. For use in experiments on induction of immuneresponse against dendritic cells loaded with melanoma cell lysate, cellgrowth was adapted to medium containing 2% human serum (HuS). Cells wereharvested by removal of medium from the culture flask and addition of 5ml of 0.02% EDTA in Ca, Mg-free PBS (BioWhittaker), incubation inCO₂-incubator for 10-20 min, addition of 10 ml of PBS and transfer ofdetached cells into a centrifuge tube. After centrifugation at 200 g for5 min, the supernatant was discarded, and the pellet was resuspended inculture medium, cells were counted, and 1.5×10⁶ cells were placed intoT75 flask in 20 ml of culture medium.

Expression of gp100 and Melan-A/MART-1 antigens was tested bydetermination of the sensitivity of melanoma cells to lysis induced bycytotoxic T lymphocyte (CTL) clones specific against these antigens. Wehave described the properties of these CTL clones previously (Kirkin etal., 1999, Cancer Immunol. Immunother., v. 48, pp. 239-246). Melanomacells were harvested as described above, resuspended in culture medium,counted, and 0.5×10⁶ cells of each clone were transferred into an 11-mlconical tube (Nunc). Cells were spun down at 200 g for 5 min, thesupernatant was discarded, and the pellet was resuspended in 0.1 ml ofculture medium. 0.1 ml of Na₂CrO₄ solution (0.1 mCi, Amersham) wasadded, and cells were incubated in a water bath at 37° C. for 60 min.After washing three times with RPMI-1640, the targets were adjusted to aconcentration at 5×10⁴ cells/ml in RPMI-1640 with 10% FCS. The cytotoxiclymphocytes were used at concentration of 5×10⁵ cells/ml. CTLs andtarget melanoma cells were seeded in 100 μl aliquots in triplicates in96 U-bottomed microtiter plates (Nunc), spun down at 200 g for 2 min andincubated at 37° C. in 5% CO₂. After 4 hours plates were spun down at250 g for 3 min, 100 μl supernatant was harvested and the radioactivitywas determined (Cobra 5005, Packard Instruments, Meriden, Conn., USA).The specific lysis was calculated according standard formula. Results ofone representative experiment are shown in FIG. 1. From these results itfollows that only two of the investigated melanoma clones, DDM-1.7 andDDM-1.13, are completely resistant to lytic attack by CTLs, indicatingpossible loss of the expression of the indicated melanocytedifferentiation antigens in these melanoma clones.

In order to get additional proof for the loss of antigen expression, weconducted analysis of expression of RNA coding for these proteins byRT-PCR analysis. 2×10⁶ cells were spun down, the supernatant wasdiscarded, and the pellet was solubilized in 0.3 ml of Cell LysisSolution (Purescript® RNA isolation kit, Gentra). RNA was isolatedaccording to the manufacturer's instructions, precipitated by adding twovolumes of 100% isopropanol over the lysis solution, washed with 70%ethanol and re-hydrated in 10 μl RNAase-free distilled water. Theisolated RNA was treated by DNAase to destroy any trace amount of DNA inthe preparation. For this purpose, the reagents from DNA-free™ kit(Ambion) were used. 1 μl 10× DNAase buffer and 1 μl DNAase (2 units)were added to the sample, the mixture was incubated for 30 min at 37°C., and reaction was terminated by addition of 1.2 μl DNAaseinactivation reagent cDNA synthesis was performed by reversetranscription in 20 μl total volume using 10 μl RNA. For this purpose,Super Script II RT primed with oligo (dT) (Gibco BRL) was used accordingto the manufacturers' protocol. Incubation was performed at 42° C. for30 min, followed by 45° C. for 30 min and 72° C. for 2 min. 1 μl cDNAwas used in the PCR amplification containing the following in 1×PCRbuffer: 50 mM KCl, 10 mM Tris/HCl (pH 9.0), 1.5 mM Mg Cl₂, 0.2 mMcresol, 12% sucrose, 0.005% bovine serum albumin, 2.5 pmol of eachprimer, 40 μM of dNTPs (Pharmacia LKB), and 1.25 U (1 μl) of AmpliTaqpolymerase (Perkin-Elmer). The primers were selected in such a way thatthe product of amplification could be efficiently cumulated at the samereaction conditions, thus, enabling us to compare expression in thetumor cells of a variety of selected antigens in simultaneouslyperformed reactions. Sequences of primers used in these experiments andin experiments described below are presented in Table 2. In allreactions, a “hot start” procedure was used in which Taq polymerase anddNTPs were added to the reaction tube at an 80° C. step between thedenaturation and annealing steps of the first cycle. The parameters usedfor the amplification were 30-38 cycles (94° C. for 30 sec, 60° C. for30 sec, and 72° C. for 40 sec) followed by 10 min at 72° C. and coolingto 4° C. Amplifications were performed on a Perkin-Elmer GeneAmp PCRSystem 9600. Negative controls contained aliquots of water instead ofcDNA. GAPDH was amplified as a positive control for the reaction as wellas to give an estimation of the rate of expression of the antigensrelative to this household gene. Negative results were repeated at leasttwice, with an increased number of cycles. The products of amplificationwere separated by electrophoresis through 2% agarose gel at 100V,stained with ethidium bromide, visualized under UV illumination andrecorded by an image recording system. When performing semi-quantitativeRT-PCR, the number of cycles was reduced to 22, which ensured a linearincrease in the amount of the selected sequences with the number ofamplification cycles. The PCR reaction was conducted using 3- or 5-folddilutions of the cDNA template. The intensity of the resulting productbands after electrophoretic separation was analyzed by image analysis,normalized by intensity of the GAPDH product obtained using the sametemplate and at the same dilutions, and the level the corresponding RNAtranscripts was compared in different cell lines.

TABLE 2 Oligonucleotide sequences 5′-3′ Gene Primer GADPH SenseAGGGGGGAGCCAAAAGGG Anti-sense GAGGAGTGGGTGTCGCTGT Gp100 SenseGGCTGGTGAAGAGACAAGTCC Anti-sense AGAGATGCAAGGACCACAGCC Mart-1 SenseGAAGGTGTCCTGTGCCCTGACCC Anti-sense GGCTTGCATTTTTCCTACACCATTCC MAGE-A1Sense GATTCCCTGGAGGCCACAG Anti-sense CCTCACTGGGTTGCCTCTGTC MAGE-A3 SenseACCAGAGGCCCCCGGAGGAG Anti-sense CTGCCAATTTCCGACGACACTCC MAGE-A4 SenseGAGCAGACAGGCCAACCG Anti-sense AAGGACTCTGCGTCAGGC MAGE-A6 SenseAGGACCAGAGGCCCCC Anti-sense GGATGATTATCAGGAAGCCTGT MAGE-A10 SenseCACAGAGCAGCACTGAAGGAG Anti-sense CTGGGTAAAGACTCAGTGTCTGG MAGE-A12 SenseTGGAAGTGGTCCGCATCG Anti-sense GCCCTCCACTGATCTTTAGCAA NY-ESO-1 SenseGGCACAGGGGGTTC Anti-sense GCTTAGCGGCCTCTGCCCT

The results of determination of expression of the melanocytedifferentiation antigens gp100 and MART-1 in 3 melanoma cell clones,DDM-1.7, DDM-1.13 and DDM-1.29, shown in FIG. 2, clearly demonstratethat the level of RNA transcripts in the DDM-1.7 and DDM-1.13 melanomaclones grown in 10% FCS is also much lower than in DDM-1.29 and theintensity of the corresponding product bands declines with serialdilutions of cDNA template much more rapidly for DDM-1.7 and DDM-1.13 ascompared to DDM-1.29. According to semi-quantitative RT-PCR and afternormalization of the data by the transcription level of housekeepingGAPDH, the level of the melanocyte antigens' RNA transcripts in DDM-1.7,DDM-1.13 was less than 1% of the corresponding level in DDM-1.29. Afteradaptation of the cells to the growth in 2% human serum, a slightincrease in the expression of the melanocyte differentiation antigens(not shown) could be seen by RT-PCR that did not exceed 1% of thecorresponding level in DDM-1.29.

Expression of gp100 in the cells adapted to the growth in 2% human serumwas also investigated by immunostaining. For this, cells have beencultured on glass cover slips placed into petri dishes. After rinsingwith cold PBS, cells have been fixed with ice-cold mixture ofmethanol-acetone (1:1) for 15 min. After drying, cover slips wereincubated in PBS for 1 min and stained according to standard procedureknown in the art using as the first antibody a mixture of antibodiesHMB45 and HMB50 (NeoMarkers), as second antibodies—biotinylated sheepanti-mouse Ig antibodies (Amersham), and as thirdreagent—streptavidin-Texas red (Amersham). As seen from FIG. 3, nostaining could be seen in DDM-1.7 cells, compared to the intensivestaining detected in DDM-1.29 cells. Visualization of a large number ofcells showed that a very small population of the cells (less than 1%)was positive in the DDM-1.7 culture (not shown). Among DDM-1.13 cellsgrown in the same conditions, about 1% of cells was positively stainedfor gp100 (not shown).

To determine levels of expression of MAGE-A and NY-ESO-1 antigens, weconducted RT-PCR reactions using primers for these antigens (sequencesof primers are presented in Table 2). The results of comparison ofexpression of mRNA coding for MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12 and NY-ESO-1 proteins are presented in FIG. 4. DDM-1.29expresses all tested antigens, while DDM-1.7 and DDM-1.13 express only4-5 of them.

Expression of CT antigens is known to be up-regulated by treatment ofcells with the DNA de-methylating agents 5-aza-2′-deoxycytidine. Wedecided to test, if expression of MAGE-A and NY-ESO-1 proteins could beup-regulated in the DDM-1.13 melanoma cell clone by such treatment.Cells were seeded in T25 culture flasks, and after 24 hours,5-aza-2′-deoxycytidine was added at final concentration of 1 μM. Cellswere incubated 3 days, then medium was changed, and after additionalincubation for two days, cells were harvested as described above.Determination of antigen expression was done as described above, and theresults, presented in FIG. 5, demonstrate that after treatment of cellswith 5-aza-2′-deoxycytidine the expression increased for all tested CTantigens.

Example 2 Generation of Dendritic Cells with Increased Abilities toPresent Exogenous Proteins

The unique property of dendritic cells is that they can presentexogenous proteins for recognition by CD8⁺ CTLs. Maximal ability touptake exogenous proteins is associated with the immature stage of DCdifferentiation. However, data on direct association between phagocyticactivity of DC and their ability to present up-taken antigens forrecognition by CTLs specific against these antigens, are absent.Therefore we conducted experiments aiming: a) to optimise generation ofimmature dendritic cells with high ability to uptake exogenous proteinsand b) to demonstrate correlation between phagocytic activity ofdendritic cells and their ability to present antigens from exogenouslyadded melanoma lysate.

In the first set of experiments we optimized generation of phagocyticdendritic cells by varying time of lymphokine addition and use ofTGF-beta 1, which was shown to improve generation of dendritic cellsfrom monocytes of peripheral blood (Yang et al., 1999, J. Immunol., v.163, pp. 1737-1741). Dendritic cells were typically generated from 50 mlof peripheral blood of HLA-A2-positive donors containing 25 IU/ml ofheparin. Blood was split in two 50-ml tubes containing 12.5 ml of Ca,Mg-free PBS (named below as PBS), and applied on Lymphoprep (12.5 mlplaced into two 50-ml tubes). After centrifugation (800 g, 25 min), 10ml of upper layer were taken into a syringe, passed through a 0.2 μmfilter and used as a source of plasma. Mononuclear cells were harvestedfrom the interphase layer, and after at least two-fold dilution withPBS, spun down 6 times, first at 650 g, 10 min, then at 450 g, 7 min,and afterwards at 250 g, 5 min. After each centrifugation, thesupernatant was discarded, and the pellet was re-suspended in 5 ml ofPBS until complete disappearance of cell aggregates. Fresh PBS was addedto fill tube to the top, and centrifugation was repeated. After lastcentrifugation, pellet was re-suspended in 5 ml of adhesion medium,consisting of RPMI 1640 medium with addition of 2% of plasma, and aftercounting, cell concentration was adjusted to 5×10⁶/ml. 3 ml of cellsuspension were placed into the wells of a 6-well plate (Falcon, non-TCtreated), totally 4 wells, and incubated in CO₂-incubator for 1.5 hours.After this incubation, non-adherent cells were collected, and monolayerof adherent cells was washed twice with warm RPMI 1640 medium, and 3 mlof culture medium consisting of RPMI medium with addition of 1% ofplasma (DC medium). Recombinant human GM-CSF and IL-4 at concentrationof 1000 U/ml were added into two wells (cultures 1 and 3). Afterovernight incubation, medium was completely changed in two wells(cultures 2 and 4). For this, medium was collected into one centrifugetube, and 2.5 ml of new pre-warmed DC medium was added into each well.Collected cells were spun down (250 g, 5 min), supernatant wasdiscarded, pellet was re-suspended in 1 ml of pre-warmed DC medium, and0.5 ml of cell suspension were placed back into cultures 2 and 4. Tocultures 3 and 4, TGF-beta 1 was added at final concentration of 100ng/ml.

After additional 5 days, phagocytic activity of generated dendriticcells was detected. RPMI 1640 medium containing 10% FCS (fetal calfserum) was placed into several wells of a flat-bottom non-TC-treated96-well plate (Falcon) as well as in centrifuge tubes for 30-60 min. 0.5ml of each DC culture was transferred into pre-treated centrifuge tubes,and after centrifugation (200 g, 5 min) the supernatant was discarded,and pellets were re-suspended in 0.5 ml of DC medium. Medium was removedfrom wells of the 96-well plate, and 0.2 ml of each cell suspension wasadded into wells, two wells for each type of culture. 10 μl of stocksolution of FluoSpheres were added to each tube, and plate was placedinto CO₂-incubator. After 4 hours, cultures were harvested intocentrifuge tubes pre-treated as described above, and after two washingsby centrifugation at 200 g, 5 min, in RPMI 1640 medium with 10% FCS,pellets were resuspended in 25 μl of RPMI medium Tubes were place intoice. 5 μl of cell suspension were place on microscope glass slides, andslides were places into moist chamber (usually large petri dishes withmoisten paper were employed) and incubated 10-15 min in CO₂-incubator.After this, a drop of cell suspension was covered by a 13-mm glass coverslip, and cells were observed under fluorescent microscope. Usingdigital camera Leica DC100, images were transferred to computer and werestored as bitmap files.

Results of one experiment on determination of phagocytic activity ofdendritic cells generated under investigated conditions are presented inFIG. 6. It is clearly seen that addition of GM-CSF and IL-4 the dayafter start of the culture together with total medium change hassignificant advantages in comparison to cultures where GM-CSF and IL-4were added from the start of cultures. It should be noted that inmajority of papers describing establishment of dendritic cells for usein immunization in combination with tumor cell lysates, lymphokines wereadded from the start of cultures (see, for example, (Chakraborty et al.,1998, Cancer Immunol. Immunother., v. 47, pp. 58-64; Nestle et al.,1998, Nature Med., v. 4, pp. 328-332)).

Results presented in FIG. 6 also demonstrate that TGF-beta 1, that wasused by investigators as additional lymphokine during generation ofdendritic cells (Yang et al., 1999, J. Immunol., v. 163, pp. 1737-1741),has no enhancing effect on phagocytic activity of dendritic cells, andin fact, decreased phagocytic activity in a number of experiments (notshown).

Ability to present antigenic peptides from exogenously added proteinswas investigated using a model of recognition of dendritic cells loadedwith lysate of DDM-1.29 cells, having high levels of expression of themelanocyte differentiation antigen gp100, by gp100-specific CTLsestablished by us (Kirkin et al., 1999, Cancer Immunol. Immunother., v.48, pp. 239-246) and used in example 1 for detection of expression ofthis antigen in different melanoma cell clones.

A lysate of DDM-1.29 cells was prepared as described below. Melanomacells cultured as described in example 1, washed twice with PBS, andre-suspended in RPMI 1640 medium (Gibco) at 10⁷ cells/ml. Cells weresubjected to five cycles of freezing (liquid nitrogen)—thawing,sonicated 15 min in a ultrasound bath (Metason 200, Struer), then spundown, first at 800 g, for 15 min at 4° C., then at 13000 g, for 60 minat 4° C. The supernatant was collected, filtrated through 0.2 μm filter,and protein concentration was determined using the bicinchoninic acidprotein assay reagent (Pierce) according to the procedure given by themanufacturer. The protein concentration was in the range of 3.5-5 mg/ml.Aliquots of supernatant were stored frozen at −80° C.

To load dendritic cells with tumor lysate, different dendritic cellcultures were transferred into centrifuge tubes, spun down, and afterdiscarding of supernatants pellets were re-suspended in 2 ml of DCmedium, cells were counted, and cell suspension was diluted up to5×10⁵/ml. 1.8 ml of cell suspension were placed into well of Falconnon-TC-treated 24-well plate, and 0.2 ml of tumor lysate were addedtogether with GM-CSF and IL-4 (1000 U/ml of each). After overnightincubation, 20 ng/ml of TNF-alfa were added. After additional incubationfor 24 hours, cultures were harvested by intensive pipetting,transferred into pre-treated centrifuge tubes, spun down at 200 g, for 5min, and supernatants were decanted, and pellets were resuspended in 2ml of DC medium. After counting, the cell suspension was diluted to3×10⁵ cells/ml, and 1 ml of cell suspension was placed in wells of24-well TC plate (Nunc), two wells for each type of dendritic cellsculture. To one of the wells 1 ml of medium was added, and to another 1ml of CTL suspension (10⁶/ml). Cultures were incubated for 24 hours,after which 1 ml of supernatant was transferred into an eppendorf tube,spun down, and the supernatant was transferred into another eppendorftube, and analysis of amount of produced interferon gamma (IFN-γ) wasperformed by ELISA method as follows. Immunoplate MaxiSorp plates (Nunc)were coated with 100 μl of anti-human IFN-γ purified monoclonal antibody(Endogen) diluted to 2 μg/ml in coating buffer (PBS, pH 7.4) byincubating overnight at room temperature. The coating solution wasremoved, 200 μl of blocking solution (4% BSA in PBS) was added, andincubated for 1 hour at room temperature. Plates were washed four timeswith PBS supplemented with 0.05% Tween-20 (washing buffer), and 50 μl oftwo-fold standard dilutions of the standard (recombinant IFN-γ, Endogen)in the culture medium, ranging from 15 to 1000 pg/ml, were added induplicate. The collected supernatants were centrifuged at 3,000 g for 5min, and 50 μl of the samples and two two-fold dilutions applied intriplicate. Plates were incubated overnight at 4° C. Without washing theplate, 50 μl of biotin-labelled detecting anti-body (anti-IFN-γbiotin-labelled, Endogen) diluted to 0.5 μg/ml in the blocking solutionwas added, and incubation continued for 2 hours at room temperature.After four-fold washing with the washing buffer, 100 μl/well ofHRP-conjugated streptavidin (Genzyme) 1:1000 diluted in the blockingbuffer was added and the plates incubated for 1 hour at roomtemperature. The plates were washed four times with the wash buffer andblotted on a paper towel. 100 μl of the substrate solution (5 mg OPD wasdissolved in 11 ml citrate buffer and supplemented with 5 μl of hydrogenperoxide) was added to each well. The reaction developed during 15-40min at room temperature and was terminated by adding 50 μl of 10%sulphuric acid. The differential absorbance was measured on an ELISAreader as a difference between the values at 490 and 650 nm. The controlvalues where only cell medium was added instead of IFN-γ, weresubtracted, and concentration of the released in the experiment IFN-γwas determined and expressed in pg/ml using the IFN-γ calibration curveplotted from the same experiment.

FIG. 7 presents data of one of these experiments. It could be seen thatmaximal specific production of IFN-γ, representing difference betweenIFN-γ production in the presence of lysate-loaded dendritic cells and“empty” dendritic cells, is maximal for dendritic cell cultures 1 and 3,the same cultures that have maximal phagocytic activity.

In summary, the ability of dendritic cells to specifically stimulateCTLs with antigens uptaken from tumor cell lysate correlates withphagocytic activity of dendritic cells, is independent of the presenceof TNF-α during DC differentiation, and is maximal in DC cultures inwhich addition of GM-CSF and IL-4 was delayed for 1 day and wasassociated with a complete medium change.

Example 3 Development of Cytotoxic T Lymphocytes with Broad Anti-TumorActivity after Stimulation of Peripheral Blood Lymphocytes of NormalDonors with Autologous Dendritic Cells Loaded with Melanoma Cell-DerivedLysate

The ability of dendritic cells loaded with melanoma cell lysate tostimulate development of cytotoxic T lymphocytes specific against tumorantigens was tested in vitro in mixed lymphocyte dendritic cell culture.Dendritic cells were generated from peripheral blood of normalHLA-A2-positive donors as described in Example 2. After initial step ofadsorption of monocytes, non-adsorbed lymphocytes were collected andfrozen down in autologous plasma plus 10% DMSO for later use. Dendriticcells loaded with tumor lysate were harvested, irradiated (6000 Rad),washed, re-suspended in X-VIVO 15 medium supplemented with 1% ofautologous plasma (complete medium) at 3×10⁵/ml and placed into fivewells of 24-well plates, 1 ml into each well. The rest of the dendriticcells was frozen in pooled human serum containing 10% DMSO. Frozenautologous non-adherent lymphocytes were thawed, washed once, counted,and 15×10⁶ cells were re-suspended after additional washing in 5 ml ofcomplete medium containing IL-7 (20 ng/ml) and IL-12 (100 pg/ml). 1 mlof lymphocyte suspension was added into wells with dendritic cells.After 7 days, 1 ml of medium was removed, and 1 ml of fresh mediumcontaining IL-7 (20 ng/ml) was added. After 5 days cells were harvested,live cells were separated on Lymphoprep (Nycomed, Norway), and afterwashing re-suspended in complete medium at 1.5×10⁶/ml. 1 ml oflymphocyte suspension was placed into wells of 24-well plates. Frozenirradiated dendritic cells loaded with lysate were thawed, washed once,resuspended in complete medium at 10s/ml, and 1 ml was added into wellswith lymphocytes. After 2 days, 1 ml of medium was removed and 1 ml offresh medium containing IL-2 (20 IU/ml) was added. This procedure ofre-stimulation was repeated every week, 2-4 times for each culture.

The lytic activity of lymphocytes was determined after 3-5 rounds ofstimulations. At this time, proliferating lymphocytes represent nearly apure population of CD3-positive cells with an increasing proportion ofCD8⁺ cells after each re-stimulation that reaches 70-90% in one weekafter 4-5^(th) round of stimulation (phenotype of cells was determinedby FACS analysis known in the art using antibody specific againstcertain surface markers).

In addition to the melanoma cell cultures mentioned above, the followingmelanoma cell lines were employed: FM28, FM55p, FM60 (HLA-A2-positive),FM45 and FM48 (HLA-A2-negative). These cell lines have been describedelsewhere (Bartkova et al, 1996, Cancer Res., v. 56, pp. 5475-5483;Kirkin et al., 1995, Cancer Immunol. Immunother., v. 41, pp. 71-81). TheDDB-1 and ANBI-EBV are EBV-transformed lymphoblastoid cell linesestablished in our laboratory by standard, well known in the artmethods. K562 erythroleukemic cells were used as target for NK-mediatedlysis. The breast cancer cell lines MCF-7, CAMA-1, HBL-100, MDA-MB-231(HLA-A2-positive) and BT-20 (HLA-A2-negative) were a kind gift from Dr.Per Briand, Danish Cancer Society. The SCC4 and SCC9 HLA-A2-positivesquamous cell carcinoma cell lines were obtained from ATCC. All celllines were cultured in RPMI 1640 medium supplemented with 10% FCS.Cytotoxic activity was determined as described in Example 1. Inexperiments with blocking antibodies, the W6/32 monoclonal antibody,specific against common determinant of HLA class I molecules, was addedinto wells at concentration of 10 μg/ml.

In the first experiments cytotoxicity was tested against panel ofmelanoma cells, as well as against EBV-transformed B cells and K562erythroleukemic cells. NK-like lytic activity, as determined by lysis ofK562 cells, was significant after two rounds of stimulations, but wasdecreased gradually after subsequent stimulations. In order to detectprimarily specific CTL-mediated lysis, all experiments on lytic activitywere performed in the presence of 20-fold excess of unlabelled K562cells. The results of the representative experiment on cytotoxicity oflymphocytes from donor ANBI after 4 rounds of stimulations withdendritic cells loaded with lysate of DDM-1.13 cells are shown in FIG.8. HLA-A2-positive melanoma cells were lysed to different degrees, whileK562, ANBI-EBV (EBV-B cells established from the same donor), DDB-1(autologous to DDM-1 melanoma cells), melanoma cell lines FM45 and FM48(having no common MHC class I antigens with cells of donor ANBI) wererelatively resistant to lysis. It is of note that FM9, HLA-A2-negativemelanoma cell line but sharing with donor ANBI HLA-A1 (not shown) isalso sensitive to lysis, indicating that HLA restriction of lysis iscomplex and not restricted by only HLA-A2 antigens. The use of untreateddendritic cells also induced lymphocyte proliferation, albeit at a lowerintensity, but development of only nonspecific cytotoxicity was seen insuch cultures (not shown). The lack of cytotoxicity against DDB-1 cells,generated from PBMCs (peripheral blood monocytes) of melanoma patientfrom which DDM-1 melanoma cells have been established, indicates thatalloantigens possibly present in the lysate preparation do not induce asignificant immune response, and that the resulting immune response ismainly tumor-specific. The W6/32 antibody against MHC class I moleculesignificantly inhibited, indicating the MHC class I-restricted nature ofcytotoxicity. Similar results were obtained with lysate isolated fromthe DDM-1.7 melanoma clone (not shown).

To test possibility that recognized antigens belong to a group ofcancer/testis antigens, shared between different types of humanmalignancies, we investigated sensitivity of a number of breast andsquamous cell carcinoma lines to lysis by generated CTLs. Results of oneexperiment are shown in FIG. 9. Three of four HLA-A2-positive breastcancer lines have moderate to high sensitivity to lysis, while oneHLA-A2-negative line was completely resistant to lysis. One of twoinvestigated squamous cell carcinoma cell lines was also sensitive tolysis. Lysis of breast cancer cell lines was sensitive to inhibition byHLA class I-specific antibody W6/32. These data demonstrate that invitro immunization of PBLs (peripheral blood lymphocytes) of a normaldonor with autologous dendritic cells loaded with lysate of DDM-1.7 orDDM-1.13 melanoma cell lines induced generation of CTLs thatspecifically recognized tumor-associated antigens present in severaltypes of human tumors.

One possible group of antigens that could be recognized by CTLsgenerated in this way is MAGE-A antigens. To correlate the expression ofthese antigens with the sensitivity of target cells to generated CTLs,we compared levels of expressions of these antigens in three breastcancer lines that show different sensitivity to lysis. Results arepresented in FIG. 10. Maximal number of investigated antigens isexpressed in HBL-100 cells, while minimal number in MCF-7 cells. Theseresults demonstrate that there is indeed a correlation between number ofthe expressed genes of MAGE-A groups and sensitivity to lysis, pointingto possibility that these or similarly regulated antigens are majortargets upon this type of immunization.

Example 4 Test for Immunodominance of Differentiation Antigens

Two melanoma cell lines expressing high levels of differentiationantigens, DDM-1.29, and low levels of differentiation antigens, DDM-1.P(variant of DDM-1 cell line) were employed in the experiments on thegeneration of cytotoxic T lymphocytes by in vitro stimulation ofperipheral blood lymphocytes with autologous tumor cells according tomethod described by Hérin et al., 1987, Int. J. Cancer, v. 39, pp.390-396. After 3 rounds of weekly stimulations of lymphocytes withirradiated tumor cells (10,000 Rad), cell cultures were cloned bylimiting dilutions, and lytic activity of growing clones was firsttested against original melanoma cell line in a Cr⁵¹-release test. Cellclones were considered cytotoxic if they had more then 20% lyticactivity against original melanoma cell line. The specificity of thecytotoxic T cell clones was then tested in the experiments with T2 cellsloaded with different HLA-A2-restricted peptides from the antigens gp100and MART-1. T2 cells untreated with peptides served as control. Around100 clones were analyzed for each melanoma cell line. Lysates wereloaded on dendritic cells as previously described and autologouslymphocytes were immunized. The proportion of CTL-clones recognizingdifferentiation antigens gp100 and MART-1 upon immunization wascalculated and the results are shown in table 3 below.

TABLE 3 Proportion (% of the total number of generated clones)of the CTLclones recognizing differentiation antigens. Proportion (% of the totalExpression of number of generated clones) of Melanoma differentiationCTL clones recognizing cell line antigens Gp100 MART-1 DDM-1.29 High 5144 DDM-1.P Low (less then 5% of 0 0 levels in DDM-1.29)

The data shows that differentiation antigens are immunodominant. In caseof high levels of expression of differentiation antigens, the immuneresponse is directed mainly towards the differentiation antigens gp100and MART-1, while no induction of immune response is seen when a cellline expressing low levels of differentiation antigens is used forimmunization. It should be noted that both cell lines induce intensiveproliferation of autologous lymphocytes with generation of large numbersof cytotoxic T cell clones.

Example 5 Phenotype of Dendritic Cells Generated at Different CultureConditions

In order to investigate how different regimes of lymphokine additioncombined with medium change at day 1, would influence the phenotypicproperties of the generated immature dendritic cells, four differentgrowth conditions were investigated. Dendritic cells were generatedessentially as described in the example 2. The different conditionswere:

1) cytokines (GM-CSF and IL-4) were added at day 0 immediately aftertermination of the adsorption step, and an additional supply ofcytokines was added at day 1 without medium change;

2) like group 1, but with a complete medium change at day 1 withadditional supply of cytokines;

3) addition of cytokines was delayed until day 1 without medium changeat day 1; and

4) the same as group 3, but with a complete medium change at day 1.

The conditions in group 4 correspond to the conditions described byThurner et al. (1999) for the generation of mature dendritic cells froma leukapheresis product (according to Thurner their optimisation shouldwork only for dendritic cells generated from leukapheresis products, butnot from freshly drawn blood samples).

After 5 days of incubation, cells were harvested and the number of largecells was counted on a Coulter Counter (Beckman, model Z2), and thecells were frozen in autologous plasma with the addition of 10% DMSO forfurther analysis of the expression of surface marker by FACS. Thefollowing monoclonal antibodies were used (all from BD Biosciences):CD1a-PE, CD14-FITC, and CD83-PE, with matching control antibodies.

Cell counts demonstrate no difference in the cell yield (in number andsize), which is in good correspondence to the data of Thurner et al.,1999, (J. Immunol. Methods 223: 1-15) since they only used morphologicalcriteria and the yield as indicators of growth optimisation. Incontrast, significant difference was seen in the present study, whensurface markers were measured (results of one of the experiments arepresented in Table 4).

TABLE 4 Phenotype of dendritic cells generated at different cultureconditions Start of Medium cytokine change at DC phenotype (%) additionday 1 CD1a CD14 CD83 Day 0 No 20.6 12.9 26.1 Day 0 Yes 18.3 9.0 34.2 Day1 No 51.0 15.4 24.9 Day 1 Yes 52.0 10.9 32.8

The start of cytokine addition from day 1 significantly up-regulates theexpression of the CD1a marker, while not significantly influencing theexpression of the other markers. It could also be seen, that mediumchange at day 1, as originally proposed by Thurner et al (1999), is infact not necessary, as the same effect was achieved both with andwithout medium change. A medium change at day 1 in fact significantlydecreased endocytic activity of the dendritic cells at the immaturestate. These conditions—delayed addition of cytokines for 1 day withoutany medium change were selected for further experiments.

Example 6 Optimization of Yield of Dendritic Cells Producible fromMonocytes

During the optimization described above we surprisingly discovered thatthe yield of dendritic cells obtainable from the mononuclear cellpopulation is dependent on the density of monocytes during theadsorption step. As noted earlier in the majority of published methods,the concentration of mononuclear cells rather than of monocytes is takeninto consideration when the adsorption step is performed. However,mononuclear cells represent a mixture of two populations, lymphocytesand monocytes, and the proportion of each population can varysignificantly. It is monocytes that predominantly adsorb to the plastic.

To estimate the concentration of monocytes in the population ofmononuclear cells we used a Coulter Counter for counting cell number,since this also permits observation of cell size distributions (Beckman,Model Z2). Two major populations of cells, with average sizes of 7 nm(lymphocytes) and 9 nm (monocytes) are seen upon counting, andappropriate gating permits the estimation of the proportion of these twopopulations.

We have previously found that at a seeding cell density of approximately15×10⁶ monocytes per T25 flask, adsorption of monocytes was almostcomplete (more than 90%), and therefore this cell density withvariations between 12×10⁶ and 20×10⁶ was used in our experiments.Significant variation in the yields of dendritic cells was observed, andin an attempt to understand the reason for such variation, we decided torelate the yield of dendritic cells in different experiments to theoriginal density of monocytes. Results of such analysis on five culturesgenerated from different donors clearly indicate the inverse correlationbetween the efficiency of dendritic cell generation and the density ofseeded monocytes.

An experiment was designed to evaluate this relationship, seedingmonocytes of the same donor at different densities. Mononuclear cellswere isolated from buffy coat (prepared from the blood of healthy donor)by centrifugation on a Lymphoprep gradient. After isolation of interfacecells and intensive washing in order to remove platelets, mononuclearcells were suspended in the culture medium consisting of RPMI 1640medium with addition of 1% of autologous heparinized plasma, and seededin T25 flasks (surface area of 25 cm²) in 7 ml medium per flask atdifferent amounts of mononuclear cells per flask (from 10×10⁶ to 20×10⁶of monocytes per flask). After 1 hour adsorption, non-adsorbed cellswere removed by washing twice with pre-warmed culture medium, and 7 mlof fresh medium were added into each flask. The next day, GM-CSF (100ng/ml) and IL-4 (50 μg/ml) were added to the culture. Cytokine additionwas repeated at day 3. At day 5, half of medium was changed, and a newportion of GM-CSF and IL-4 was added. At day 6, TNF-α was added at aconcentration of 20 ng/ml. The cultures were harvested at day 7, andafter counting total number of cells they were frozen, in autologousserum with 10% of DMSO and kept until analyzed by FACS analysis. FACSanalysis was performed with CD1a and CD14 antibodies.

The results of one of the experiments regarding the yield of dendriticcells (large cells with the median size between 14 and 16 nm) arepresented in the Table 5.

TABLE 5 Yield and phenotype of dendritic cells in cultures withdifferent initial density of the monocytes No. of monocytes No. of Yieldof per T25 DC's per DC's, DC phenotype, % flask T25 flask % of of gatedcells (×10⁶) (×10⁶) monocytes CD1a CD14 10 5.28 52.8 54.9 11.0 12.5 5.4343.4 44.9 16.9 15 4.67 31.1 46.4 14.9 17.5 4.47 25.5 46.2 14.5 20 4.6923.4 44.2 7.4

The yield of dendritic cells was significantly decreased with increasingdensity of the seeded monocytes, being 50% at minimal cell density. Theresults of a FACS analysis performed on the same dendritic cells arealso presented in the table and indicate that no significant differencewas observed in the properties of dendritic cells generated underinitially different density of the monocytes.

Decreasing the initial cell density of monocytes below 10×10⁶ cells perT25 flask leads to a decrease in the expression of the CD1a marker (datanot shown). Therefore, the optimal cell density of initial monocytepopulation leading to the maximal efficiency of the production ofdendritic cells with maximal competence (as judged by the expression ofthe major DC markers) is around 10×10⁶ cells per 25 cm2 of culturesurface.

Based on these data, it is now possible to calculate the volume of bloodthat will be necessary to draw. If the expected yield of dendritic cellsis about 50% of the initial number of monocytes, then it will requireonly 300 ml of blood to obtain 50×10⁶ dendritic cells. This means, thatone single blood sample of 300 ml of blood should be enough for thepreparation of a vaccine for two rounds of vaccinations. The requiredamount of cytokines (21 ng for GM-CSF and 10.5 ng for IL-4), as well asother materials is three times lower than in the standard, non-optimizedmethod. Therefore, also the cost of materials needed for the DCproduction will be at least three times lower, and in addition therewill be no need for conducting expensive leukapheresis procedures.

Example 7 Use of Dendritic Cell Based Vaccine

A vaccination cycle of cancer patients with dendritic cells loaded withthe lysate of two melanoma cell lines prepared according to theinvention, should preferably be designed in such a way that patientswill receive a first cycle of five intradermal vaccinations with 3 weekintervals, each time with 5×10⁶ dendritic cells. If a clinical responseis seen after the initial cycle of vaccinations, a second, similar cyclewill be conducted. For the whole period of the trial a total of up to50×10⁶ cells will be required. Considering the condition of the patients(the majority of patients are at advanced stages of disease, where noother treatments are working anymore), it is important to minimize thenumbers of blood drawings, restricting them to one or two.

1-62. (canceled)
 63. A melanoma cell line DDM-1.7 deposited an ECACC01112339.
 64. A melanoma cell line DDM1-1.13 deposited an ECACC01112338.
 65. Exosomes obtained from a cell line DDM-1.7 (ECACC01112339) or DDM-1.13 (ECACC 01112338).